Cyclic amino acid molecules and methods of preparing the same
10385099 · 2019-08-20
Assignee
Inventors
Cpc classification
C07K1/006
CHEMISTRY; METALLURGY
C07K7/64
CHEMISTRY; METALLURGY
C07K1/003
CHEMISTRY; METALLURGY
International classification
C07K7/64
CHEMISTRY; METALLURGY
C07K5/12
CHEMISTRY; METALLURGY
C07F7/18
CHEMISTRY; METALLURGY
Abstract
Macrocyclization of amino acids or linear peptides upon reaction with amphoteric amino aldehydes and isocyanides is provided.
Claims
1. A cyclic compound of formula (III) ##STR00028## wherein n=0 or 1, and R1, R2, R3, R4 and R5 are independently selected from H; lower alkyl; aryl; heteroaryl; alkenyl; heterocycle; cyckoalkyl; esters of the formula C(O)OR* wherein R* is selected from alkyl and aryl; amides of the formula C(O)NR**R***, wherein R** and R*** are independently selected from alkyl and aryl; CH.sub.2C(O)R, wherein R is selected from OH, lower alkyl, aryl, -lower alkyl-aryl, or NR.sub.aR.sub.b, where R.sub.a and R.sub.b are independently selected from H, lower alkyl, aryl or -lower alkyl-aryl; C(O)R.sub.c, wherein R.sub.c is selected from lower alkyl, aryl or -lower alkyl-aryl; or -lower alkyl-OR.sub.d, wherein R.sub.d is a suitable protecting group or OH group; bonds [a] and [b] are syn or antito each other; R is an amino acid side chain of the amino terminus amino acid; R is an optionally substituted amide; and R.sup.6 is a nucleophilic moiety of a compound having the formula R.sup.6H, wherein R.sup.6H is selected from the group consisting of 7-mercapto-4-methyl-coumarin, thiobenzoic acid, thioacetic acid, biotin, aromatic thiols, aliphatic thiols; and is capable of nucleophilic opening of the aziridine ring of a compound of formula (II) ##STR00029## to form the compound of formula (III); and the linear molecule is an amino acid, a linear peptide comprising 2-9 amino acids, or a salt of the amino acid or the linear peptide, wherein N is the nitrogen at the amino terminus end of the linear molecule and C is the carbon at the carboxy terminus end of the linear molecule, and provided that if the linear molecule is a linear peptide, bonds [a] and [c] are antito each other.
2. The cyclic compound of claim 1, wherein R.sup.6 is a fluorescent moiety.
3. The cyclic compound of claim 1, wherein any one of R.sub.1-R5 is H.
4. The cyclic compound of claim 1, wherein n=0 and R.sub.1-R.sub.3 are H.
5. The cyclic compound of claim 3, wherein n=0 and R.sub.2 and R.sub.3 are H.
6. The cyclic compound of claim 1, wherein R.sub.1 is CH.sub.2OTBDMS or CH.sub.2.sup.iPr.
7. The cyclic compound of claim 1 wherein the linear molecule is a peptide.
8. The cyclic compound of claim 1, wherein the amino terminus amino acid of the peptide is selected from the group consisting of proline and an amino acid with an amino group substituted with NHCH.sub.3, NHBn, NHCH.sub.2CH.sub.2SO.sub.2Ph or NHCH.sub.2CH.sub.2CN.
9. The cyclic compound of claim 1, wherein the linear molecule is a D- or L-amino acid selected from the group consisting of: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, selenocysteine, serine, tyrosine, threonine, tryptophan and valine.
10. The cyclic compound of claim 1, wherein the linear molecule is an alpha-amino acid.
11. The cyclic compound of claim 1, wherein the linear molecule is a beta-amino acid.
12. The cyclic compound of claim 1, wherein the linear molecule is a gamma-amino acid.
13. The cyclic compound of claim 1, wherein R is tert-Butyl amide.
14. The cyclic compound of claim 1, wherein the linear peptide consists of 2 amino acids.
15. The cyclic compound of claim 1, wherein the linear peptide consists of 3 amino acids.
16. The cyclic compound of claim 1, wherein the linear peptide consists of 4 amino acids.
17. The cyclic compound of claim 1, wherein the linear peptide consists of 5 amino acids.
18. The cyclic compound of claim 1, wherein the linear peptide consists of 6 amino acids.
19. The cyclic compound of claim 1, wherein the linear peptide consists of 7 amino acids.
20. The cyclic compound of claim 1, wherein the linear peptide consists of 8 amino acids.
21. The cyclic compound of claim 1, wherein the linear peptide consists of 9 amino acids.
22. The cyclic compound of claim 1, wherein the nucleophilic moiety is bonded to XH, wherein X is S.
23. The cyclic compound of claim 1, wherein R6-H is 7-mercapto-4-methyl-coumarin or thiobenzoic acid.
24. The cyclic compound of claim 1, wherein if the amino acid molecule is an amino acid then the amino terminus is a primary amino group or a secondary amino group but wherein the amino acid molecule is a linear peptide, then the amino terminus is a secondary amino group.
Description
BRIEF DESCRIPTION OF THE FIGURES
(1) Embodiments of the invention may best be understood by referring to the following description and accompanying drawings. In the description and drawings, like numerals refer to like structures or processes. In the drawings:
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DETAILED DESCRIPTION
(34) There is described herein a one-step process that provides cyclic peptides in high yields and selectivities while evading the problems typically encountered during traditional cyclization reactions. The late stage diversification of macrocyclic molecules can now be achieved in a seamless fashion. The products of the macrocyclization are equipped with specific modification sites, which enable late-stage structural modification of cyclic peptides. The post cyclization diversification has been a historic challenge for macrocycle libraries in both biotechnology and pharmaceutical industries. The present macrocyclization approach solves this problem, among others.
(35) There is demonstrated herein a use for a class of stable amphoteric amino aldehydes that contain synthetically useful unprotected aziridine and aldehyde groups..sup.8 Amphoteric amino aldehydes are coaxed into undergoing a reversible reaction with the amine and carboxyl ends of the peptide. The resulting electrophilic intermediate rapidly reacts with the nucleophilic aziridine portion due to the high effective molarity. As a result, the unfavorable chain/ring equilibrium is shifted using the Le Chatelier principle, securing efficient conversion to cyclic products. In another aspect, the cyclization tolerates aliphatic, acidic, and basic amino acid side chain residues. In particular, peptides, including those containing both acidic and basic residues, are found to be within the scope of this process, delivering a single diastereomeric product in each case. Trifluoroethanol (TFE), known for its capacity to stabilize peptide secondary structure and promote polar interactions,.sup.9 is the preferred reaction medium. The Ugi four-component condensation, a well-known reaction involving carboxylic acids, amines, aldehydes, and isocyanides.sup.10, is the preferred means of generating zwitterionic macrocyclization precursors. Mechanistically, the Ugi reaction is known to proceed through a series of reversible transformations that tend towards the thermodynamic driving force of amide bond formation. When an -amino acid, aldehyde, and isocyanide are used as starting materials, the reaction traverses a zwitterionic iminium ion intermediate, which upon attack by the isocyanide produces an electrophilic mixed anhydride. Subsequent reaction with methanol gives a linear peptide ester as a mixture of diastereoisomers..sup.11 This reaction has been attempted in the synthesis of cyclic peptides from linear peptides and conventional (monofunctional) aldehydes. However, low yields are accompanied by lack of diastereoselectivity and dominant cyclodimerization products..sup.12,13
(36) In one aspect, there is provided a process to produce a cyclic amino acid molecule comprising reacting an amino acid molecule, having an amino terminus and a carboxyl terminus, with an isocyanide and a compound having formula (Ia) and/or (Ib):
(37) ##STR00005##
wherein: n=0 or 1, and R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are independently selected from H; lower alkyl; alkenyl; heterocycle; cyckoalkyl; esters of the formula C(O)OR* wherein R* is selected from alkyl and aryl; amides of the formula C(O)NR**R***, wherein R** and R*** are independently selected from alkyl and aryl; CH.sub.2C(O)R, wherein R is selected from OH, lower alkyl, aryl, -lower alkyl-aryl, or NR.sub.aR.sub.b, where R.sub.a and R.sub.b are independently selected from H, lower alkyl, aryl or -lower alkyl-aryl; C(O)R.sub.c, wherein R.sub.c is selected from lower alkyl, aryl or -lower alkyl-aryl; or -lower alkyl-OR.sub.d, wherein R.sub.d is a suitable protecting group or OH group; all of which are optionally substituted at one or more substitutable positions with one or more suitable substituents; and the aldehyde component thereof may optionally be in its bisulfite adduct form; and the amino acid molecule is an amino acid, a linear peptide or a salt of the foregoing, provided that if the amino acid molecule is a linear peptide, the compound comprises an aziridine chiral center proximal to the aldehyde with matching stereochemistry to the carbon atom proximal to the amino terminus of the peptide.
(38) Preferably, if the amino acid molecule is an amino acid then the amino terminus is a primary amino group or a secondary amino group but wherein the amino acid molecule is a peptide, then the amino terminus is a secondary amino group.
(39) In one embodiment, any one of R.sub.1-R.sub.5 is H. Preferably, n=0 and R.sub.2 and R.sub.3 is H or R.sub.1-R.sub.3 is H.
(40) In a particular embodiment R.sub.1 is CH.sub.2OTBDMS or CH.sub.2.sup.iPr.
(41) In one embodiment, the amino acid molecule is a linear peptide. Preferably, the amino terminus amino acid of the linear peptide is selected from the group consisting of proline and an amino acid with an amino group substituted with NHBn, NHCH.sub.2CH.sub.2SO.sub.2Ph or NHCH.sub.2CH.sub.2CN.
(42) In another embodiment, the amino acid molecule is a D or L amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, selenocysteine, serine, tyrosine, threonine, tryptophan and valine.
(43) The amino acid molecule may be an alpha-amino acid, beta-amino acid or gamma-amino acid.
(44) In one embodiment the isocyanide is selected from the group consisting of: (S)-()--Methylbenzyl isocyanide; 1,1,3,3-Tetramethylbutyl isocyanide; 1-Pentyl isocyanide; 2,6-Dimethylphenyl isocyanide; 2-Morpholinoethyl isocyanide; 2-Naphthyl isocyanide; 2-Pentyl isocyanide; 4-Methoxyphenyl isocyanide; Benzyl isocyanide; Cutyl isocyanide; Cyclohexyl isocyanide; Isopropyl isocyanide; p-Toluenesulfonylmethyl isocyanide; Phenyl isocyanide dichloride; tert-Butyl isocyanide; (Trimethylsilyl)methyl isocyanide; 1H-Benzotriazol-1-ylmethyl isocyanide; 2-Chloro-6-methylphenyl isocyanide; Di-tert-butyl 2-isocyanosuccinate; tert-Butyl 2-isocyano-3-methylbutyrate; tert-Butyl 2-isocyano-3-phenylpropionate; tert-Butyl 2-isocyanopropionate; and tert-Butyl 3-isocyanopropionate, preferably, tert-Butyl isocyanide.
(45) In some embodiments, the process is conducted in a non-nucleophilic reaction medium, preferably trifluoroethanol or HFIP mixed with water.
(46) In one embodiment, if the amino acid molecule is an amino acid, the process is conducted in water.
(47) In some embodiments, the process further comprises conjugating a fluorescent tag to the cyclic amino acid molecule by nucleophilic ring-opening of the aziridine moiety.
(48) In some embodiments, the peptide is between 2 and 30 amino acids in length.
(49) In another aspect, there is provided a cyclic amino acid molecule prepared using the process described herein.
(50) One would understand that cyclization, in some cases, may require and would include protecting certain peptide or amino acid side chains in manner known to a person skilled in the art.
(51) In another aspect, there is provided a cyclic amino acid molecule of formula (II):
(52) ##STR00006##
wherein, n=0 or 1, and R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are independently selected from H; lower alkyl; aryl; heteroaryl; alkenyl; heterocycle; esters of the formula C(O)OR* wherein R* is selected from alkyl and aryl; amides of the formula C(O)NR**R***, wherein R** and R*** are independently selected from alkyl and aryl; CH.sub.2C(O)R, wherein R is selected from OH, lower alkyl, aryl, -loweralkyl-aryl, or NR.sub.aR.sub.b, where R.sub.a and R.sub.b are independently selected from H, lower alkyl, aryl or -loweralkyl-aryl; C(O)R.sub.c, wherein R.sub.c is selected from lower alkyl, aryl or -lower alkyl-aryl; or -lower alkyl-OR.sub.d, wherein R.sub.d is a suitable protecting group or OH group; all of which are optionally substituted at one or more substitutable positions with one or more suitable substituents, bonds [a] and [b] are syn to each other; R is an amino acid side chain of the amino terminus amino acid; R is an optionally substituted amide; and the amino acid molecule is an amino acid or a linear peptide, wherein N is the nitrogen at the amino terminus end of the amino acid molecule and C is the carbon at the carboxy terminus end of the amino acid molecule, and provided that if the amino acid molecule is a linear peptide, bonds [a] and [c] are anti to each other.
(53) Preferably, if the amino acid molecule is an amino acid then the amino terminus is a primary amino group or a secondary amino group but wherein the amino acid molecule is a linear peptide, then the amino terminus is a secondary amino group.
(54) In one embodiment, any one of R.sub.1-R.sub.5 is H. Preferably, n=0 and R.sub.2 and R.sub.3 is H or R.sub.1-R.sub.3 is H.
(55) In a particular embodiment R.sub.1 is CH.sub.2OTBDMS or CH.sub.2.sup.iPr.
(56) In one embodiment, the amino acid molecule is a linear peptide. Preferably, the amino terminus amino acid of the linear peptide is selected from the group consisting of proline and an amino acid with an amino group substituted with NHBn, NHCH.sub.2CH.sub.2SO.sub.2Ph or NHCH.sub.2CH.sub.2CN.
(57) In another embodiment, the amino acid molecule is a D or L amino acid selected from the group consisting of: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, selenocysteine, serine, tyrosine, threonine, tryptophan and valine.
(58) The amino acid molecule may be an alpha-amino acid, beta-amino acid or gamma-amino acid.
(59) In one embodiment R is tert-Butyl amide.
(60) In another aspect, there is provided use of a compound for cyclizing an amino acid molecule, wherein the compound has the formula (Ia)/(Ib):
(61) ##STR00007## comprises an -stereocenter at the carbon atom proximal to the aldehyde group with matching stereochemistry to the carbon atom proximal to the amino terminus of the amino acid molecule; and n=0 or 1, and R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are independently selected from H; lower alkyl; aryl; heteroaryl; alkenyl; heterocycle; esters of the formula C(O)OR* wherein R* is selected from alkyl and aryl; amides of the formula C(O)NR**R***, wherein R** and R*** are independently selected from alkyl and aryl; CH.sub.2C(O)R, wherein R is selected from OH, lower alkyl, aryl, -loweralkyl-aryl, or NR.sub.aR.sub.b, where R.sub.a and R.sub.b are independently selected from H, lower alkyl, aryl or -loweralkyl-aryl; C(O)R.sub.c, wherein R.sub.c is selected from lower alkyl, aryl or -loweralkyl-aryl; or -lower alkyl-OR.sub.d, wherein R.sub.d is a suitable protecting group; all of which are optionally substituted at one or more substitutable positions with one or more suitable substituents; and the amino acid molecule is an amino acid, a linear peptide or a salt of the foregoing, provided that if the amino acid molecule is a linear peptide, the compound comprises an aziridine chiral center proximal to the aldehyde with matching stereochemistry to the carbon atom proximal to the amino terminus of the peptide.
(62) As used herein, the term amino acid molecule is meant to include single amino acids and also peptides.
(63) As used herein, the term amino acid refers to molecules containing an amine group, a carboxylic acid group and a side chain that varies. Amino acid is meant to include not only the twenty amino acids commonly found in proteins but also non-standard amino acids and unnatural amino acid derivatives known to those of skill in the art, and therefore includes, but is not limited to, alpha, beta and gamma amino acids. Peptides are polymers of at least two amino acids and may include standard, non-standard, and unnatural amino acids.
(64) The term suitable substituent as used in the context of the present invention is meant to include independently H; hydroxyl; cyano; alkyl, such as lower alkyl, such as methyl, ethyl, propyl, n-butyl, t-butyl, hexyl and the like; alkoxy, such as lower alkoxy such as methoxy, ethoxy, and the like; aryloxy, such as phenoxy and the like; vinyl; alkenyl, such as hexenyl and the like; alkynyl; formyl; haloalkyl, such as lower haloalkyl which includes CF.sub.3, CCl.sub.3 and the like; halide; aryl, such as phenyl and napthyl; heteroaryl, such as thienyl and furanyl and the like; amide such as C(O)NR.sub.aR.sub.b, where R.sub.a and R.sub.b are independently selected from lower alkyl, aryl or benzyl, and the like; acyl, such as C(O)C.sub.6H.sub.5, and the like; ester such as C(O)OCH.sub.3 the like; ethers and thioethers, such as O-Bn and the like; thioalkoxy; phosphino; and NR.sub.aR.sub.b, where R.sub.a and R.sub.b are independently selected from lower alkyl, aryl or benzyl, and the like. It is to be understood that a suitable substituent as used in the context of the present invention is meant to denote a substituent that does not interfere with the formation of the desired product by the processes of the present invention.
(65) As used in the context of the present invention, the term lower alkyl as used herein either alone or in combination with another substituent means acyclic, straight or branched chain alkyl substituent containing from one to six carbons and includes for example, methyl, ethyl, 1-methylethyl, 1-methylpropyl, 2-methylpropyl, and the like. A similar use of the term is to be understood for lower alkoxy, lower thioalkyl, lower alkenyl and the like in respect of the number of carbon atoms. For example, lower alkoxy as used herein includes methoxy, ethoxy, t-butoxy.
(66) The term alkyl encompasses lower alkyl, and also includes alkyl groups having more than six carbon atoms, such as, for example, acyclic, straight or branched chain alkyl substituents having seven to ten carbon atoms.
(67) The term aryl as used herein, either alone or in combination with another substituent, means an aromatic monocyclic system or an aromatic polycyclic system. For example, the term aryl includes a phenyl or a napthyl ring, and may also include larger aromatic polycyclic systems, such as fluorescent (eg. anthracene) or radioactive labels and their derivatives.
(68) The term heteroaryl as used herein, either alone or in combination with another substituent means a 5, 6, or 7-membered unsaturated heterocycle containing from one to 4 heteroatoms selected from nitrogen, oxygen, and sulphur and which form an aromatic system. The term heteroaryl also includes a polycyclic aromatic system comprising a 5, 6, or 7-membered unsaturated heterocycle containing from one to 4 heteroatoms selected from nitrogen, oxygen, and sulphur.
(69) The term cycloalkyl as used herein, either alone or in combination with another substituent, means a cycloalkyl substituent that includes for example, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
(70) The term cycloalkyl-alkyl- as used herein means an alkyl radical to which a cycloalkyl radical is directly linked; and includes, but is not limited to, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, 1-cyclopentylethyl, 2-cyclopentylethyl, cyclohexylmethyl, 1-cyclohexylethyl and 2-cyclohexylethyl. A similar use of the alkyl or lower alkyl terms is to be understood for aryl-alkyl-, aryl-loweralkyl- (eg. benzyl), -lower alkyl-alkenyl (eg. allyl), heteroaryl-alkyl-, and the like as used herein. For example, the term aryl-alkyl- means an alkyl radical, to which an aryl is bonded. Examples of aryl-alkyl- include, but are not limited to, benzyl (phenylmethyl), 1-phenylethyl, 2-phenylethyl and phenylpropyl.
(71) As used herein, the term heterocycle, either alone or in combination with another radical, means a monovalent radical derived by removal of a hydrogen from a three- to seven-membered saturated or unsaturated (including aromatic) heterocycle containing from one to four heteroatoms selected from nitrogen, oxygen and sulfur. Examples of such heterocycles include, but are not limited to, aziridine, epoxide, azetidine, pyrrolidine, tetrahydrofuran, thiazolidine, pyrrole, thiophene, hydantoin, diazepine, imidazole, isoxazole, thiazole, tetrazole, piperidine, piperazine, homopiperidine, homo-piperazine, 1,4-dioxane, 4-morpholine, 4-thiomorpholine, pyridine, pyridine-N-oxide or pyrimidine, and the like.
(72) The term alkenyl, as used herein, either alone or in combination with another radical, is intended to mean an unsaturated, acyclic straight chain radical containing two or more carbon atoms, at least two of which are bonded to each other by a double bond. Examples of such radicals include, but are not limited to, ethenyl (vinyl), 1-propenyl, 2-propenyl, and 1-butenyl.
(73) The term alkynyl, as used herein is intended to mean an unsaturated, acyclic straight chain radical containing two or more carbon atoms, at least two of which are bonded to each other by a triple bond. Examples of such radicals include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, and 1-butynyl.
(74) The term alkoxy as used herein, either alone or in combination with another radical, means the radical O(C.sub.1-n)alkyl wherein alkyl is as defined above containing 1 or more carbon atoms, and includes for example methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy and 1,1-dimethylethoxy. Where n is 1 to 6, the term lower alkoxy applies, as noted above, whereas the term alkoxy encompasses lower alkoxy as well as alkoxy groups where n is greater than 6 (for example, n=7 to 10). The term aryloxy as used herein alone or in combination with another radical means O-aryl, wherein aryl is defined as noted above.
(75) A peptide is a polymer of two or more amino acids.
(76) The following examples are illustrative of various aspects of the invention, and do not limit the broad aspects of the invention as disclosed herein.
EXAMPLES
Example 1
Materials and Methods
(77) Anhydrous toluene and dimethylformamide (DMF) were purchased and used as received. Tetrahydrofuran (THF) was distilled from sodium benzophenone ketyl under argon. All other solvents including TFE (2,2,2-trifluoroethanol) and HFIP (1,1,1,3,3,3-hexafluoro-2-isopropanol) were of reagent grade quality. Melting points were obtained on a MelTemp melting-point apparatus and are uncorrected.
(78) Method 1: amino acid/peptide cyclization reaction was conducted using the following method. In a screw-cap vial equipped with a magnetic stirring bar was added peptide (0.2 mmol) and 1 ml of TFE and stirred until a homogeneous solution has been obtained. Aziridine aldehyde dimer (0.1 mmol) and isocyanide (0.2 mmol) were then added sequentially and the resulting mixture was stirred for the time specified in Table 1. Reactions were monitored by electrospray ionization mass spectrometry (ESI-MS) at 60 eV and/or thin layer chromatography (TLC) analysis. After completion of the reaction, 1 ml of water and 1 ml of Et.sub.2O were added and the mixture was shaken vigorously and then cooled on ice. The resulting precipitate was filtered and washed with hexanes and cold Et.sub.2O (1 ml) to afford the cyclic peptide. For products that are water soluble or do not precipitate, the reaction mixture was concentrated under reduced pressure and then triturated with Et.sub.2O and hexanes (0.2 ml) to afford the cyclic peptide product.
(79) Method 2: amino acid/peptide cyclization reaction was conducted using HFIP in place of TFE (Method 2). In a screw-cap vial equipped with a magnetic stirring bar was added peptide (0.2 mmol) and 1 ml of HFIP and stirred until homogeneous solution has been obtained. Aziridine aldehyde dimer (0.1 mmol) and isocyanide (0.2 mmol) were then added sequentially and the resulting mixture was stirred for the time specified in Table 1. Reactions were monitored by ESI-MS at 60 eV and/or TLC analysis. After completion of the reaction, the mixture was concentrated under reduced pressure and then triturated with Et.sub.2O and hexanes (0.2 ml) to afford the cyclic peptide product.
(80) Method 3: amino acid/peptide cyclization reaction was carried out using HFIP and water. In a screw-cap vial equipped with a magnetic stirring bar was added peptide (0.2 mmol) and 1 ml of HFIP and 47 microliters of H.sub.2O and stirred until homogeneous. Aziridine aldehyde dimer (0.1 mmol) and isocyanide (0.2 mmol) were then added sequentially and the resulting mixture was stirred for the time specified in Table 1. Reactions were monitored by ESI-MS at 60 eV and/or TLC analysis. After completion of the reaction, the mixture was concentrated under reduced pressure and then triturated with Et.sub.2O and hexanes to afford the cyclic peptide product.
(81) Note: In reactions involving either cysteine or peptides containing thiol residues, the solvent was degassed with argon for two hours prior to reaction. The reaction was thereafter carried out under an atmosphere of argon.
(82) Some of the cyclic products that may be synthesized from each of Methods 1-3 are summarized in Table 3 and depicted in
(83) TABLE-US-00001 TABLE 3 Methods used for amino acid/peptide cyclization reactions. Method Cyclic Products 1 1, 2, 3, 4, 7, 9, 11, 12, 15, 16, 17, 18, 19, 20, 21, 22 2 5, 8, 10, 13, 14 3 6
(84) The relative stereochemistry of each cyclic product listed in Table 3 was established by correlating the methine region of each .sup.1H NMR spectrum with that of piperazinone 1.
(85) Chromatography: Flash column chromatography was carried out using Silicycle 230-400 mesh silica gel. Thin-layer chromatography (TLC) was performed on Macherey Nagel pre-coated glass backed TLC plates (SIL G/UV254, 0.25 mm) and visualized using a UV lamp (254 nm) and iodine stain.
(86) Nuclear magnetic resonance spectra: .sup.1H and .sup.13C NMR spectra were recorded on Varian Mercury 400 or 500 MHz spectrometers. .sup.1H NMR spectra were referenced to TMS (0 ppm) and .sup.13C NMR spectra were referenced to CDCl3 (77.23 ppm). Peak multiplicities are designated by the following abbreviations: s, singlet; bs, broad singlet; d, doublet; t, triplet; q, quartet; m, multiplet; ds, doublet of singlets; dd, doublet of doublets; ddd, doublet of doublet of doublets; bt, broad triplet; td, triplet of doublets; tdd, triplet of doublets of doublets.
(87) Mass Spectroscopy: High-resolution mass spectra were obtained on a VG 70-250S (double focusing) mass spectrometer at 70 eV or on an ABI/Sciex Qstar mass spectrometer with ESI source, MS/MS and accurate mass capabilities. Low resolution mass spectra (ESI) were obtained at 60 eV, 70 eV and 100 eV.
(88) Synthetic routes toward aziridine aldehyde dimers: All dimeric amino aldehydes used in this study were prepared according to the synthetic route outlined in Supplementary
(89) Results
(90) Diverse piperazinones were produced in a one-step process (Table 1).
(91) TABLE-US-00002 TABLE 1 The scope of piperazinone synthesis (L-amino acid-derived product is shown).
(92) The step that shifts the ring/chain equilibrium is transannular collapse of the nucleophilic NH aziridine onto the electrophilic mixed anhydride generated when an amphoteric amino aldehyde is subjected to the Ugi reaction condition (Scheme 2). The reaction takes place under stoichiometric conditions and delivers cyclic peptide product. High dilution, a critical condition normally required in order to achieve high yields in cyclic peptide synthesis, is not necessary.
(93) ##STR00011##
(94) The normally challenging medium-sized rings can be made with great facility and in good yields within several hours (Table 2).
(95) TABLE-US-00003 TABLE 2 Representative scope of linear peptide macrocyclization.
(96) .sup.1H and .sup.13C NMR spectra for the cyclic products of Table 3 are shown in
(97) A one-step process for making versatile cyclic peptides with high stereo- and chemoselectivity for linear peptides, isocyanides, and amphoteric amino aldehydes is provided. The Ugi reaction is run with an amphoteric amino aldehyde in place of a monofunctional aldehyde. When L-phenyl alanine was reacted with tert-butyl isocyanide, and an aziridine aldehyde, a cyclic piperazinone was obtained in 92% yield in approximately one hour (Table 1, Entry 1). The relative stereochemistry of 1 was established using X-ray analysis. A range of amino acids were subjected to this reaction and in all cases the corresponding piperazinone products were obtained as single diastereoisomers without formation of linear peptides (
(98) The existence of epimeric products were investigated through the use of .sup.1H NMR (DMSO-d.sub.6 or CDCl.sub.3) analysis of crude reaction mixtures. In all of the reactions studied, there were no detectable signals corresponding to the epimer. As such, the resulting crude cyclic peptides were obtained in >20:1 selectivity.
(99) The effect of medium-sized peptide chain length on the reaction outcome was studied (Table 2). The challenging medium-sized rings are readily prepared; the reaction times are less than 10 hours, proceeding with high yields and diastereoselectivities. The preferred work-up procedure involves product precipitation from diethyl ether and hexanes. The cyclic peptides require no further purification by HPLC. In addition, racemization was not detected throughout the course of the reaction or during product isolation. The lack of epimerization is further evidenced by high stereoselectivity; aziridine aldehydes with S stereocenters next to the carbonyl group undergo macrocyclization with the peptides that contain an L-amino acid residue at the N-terminus. The mismatched reaction with the D-amino acid-terminated peptide is unproductive, leading to the formation of stable aminals.
(100) It has been determined that the -stereocenter of the N-terminal amino acid, in this case proline, should match the -stereocenter of the aziridine aldehyde in order for cyclization to occur, with respect to peptides. In a mismatched reaction, the only product formed is the corresponding aminal (
(101) When the opposite enantiomer of amino aldehyde was used in the same reaction, the proline and the amino aldehyde both had S-configuration at their -stereocenters. This matched case readily underwent peptide cyclization to yield the corresponding cyclic product in high yield (
(102) This stereoselectivity process is significant in the context of possible epimerization at the N-terminus. Since matched stereochemistry is required for the cyclization to occur, the resulting cyclic peptides will necessarily contain matching stereocenters. Furthermore, the fact that no cyclic peptide formation was observed in mismatched reactions suggests that no epimerization, which would result in matched substrates, occurs under our macro conditions.
(103) Consistent yields in this chemistry are likely obtained due to the mechanism that governs cyclization. It is at this point that a departure from the Ugi reaction with monofunctional aldehydes becomes apparent. When monofunctional aldehydes are used in the reaction with isocyanides and peptides, low diastereoselectivities are observed. More importantly, the undesired cyclodimerization occurs during the cyclization of linear peptides containing less than six residues; the cyclization of tripeptides yields only the cyclodimers..sup.13 This low selectivity is due to a slow transannular attack of the amine onto the mixed anhydride (Scheme 2, A), thus allowing the intermolecular process to be kinetically competitive. By adding an amphoteric amino aldehyde to the cyclization reaction mixture containing a secondary amine-terminated peptide, the slow transannular attack is being replaced with a fast attack by the nucleophilic aziridine, which is positioned exocyclic to the mixed anhydride (Scheme 2, B). This provides an unencumbered trajectory of attack. Since TFE is a non-nucleophilic solvent, premature solvolysis of the mixed anhydride is not observed. The reaction mechanism ensures that the C-terminus is activated only upon formation of the intermediate cyclic mixed anhydride, which is then attacked by the exocyclic aziridine. This not only secures selectivity for the intramolecular macrocyclization, but also avoids prolonged C-terminus activation and potential epimerization. Accordingly, high dilution, a critical condition normally required in order to achieve high yields in conventional cyclic peptide synthesis, is not necessary..sup.6 Furthermore, no oligomeric or polymeric by-products were detected in the subject experiments. A side-by side comparison of the subject reaction with the traditional lactamization of a linear tetrapeptide, widely used in the synthesis of cyclic peptides, was conducted. The amino aldehyde mediated macrocyclization delivered rapid, selective, and efficient formation of the cyclic peptide. In contrast, only trace amount of the cyclic peptide was detected among myriad structures formed in the course of the lactamization. The undesired cyclodimerization.sup.5 has dominated this process, which is a testament to sluggish kinetics of the conventional intramolecular process. Peptide macrocyclization reactions are normally performed under extremely dilution. Typical protocols necessitate molar concentrations of 10.sup.4 M. This large dilution increases the selectivity for intramolecular cyclizations, thereby limiting undesired cyclodimerization and trimerization.
(104) Characterization of the relative stereochemistry of each cyclic product listed in Table 3 is set out below.
(3S,5R,6R,7S)-3-benzyl-N-tert-butyl-7-((tert-butyldimethylsilyloxy)methyl)-2-oxo-1,4-diazabicyclo[4.1.0]heptane-5-carboxamide (1)
(105) .sup.1H NMR (CDCl.sub.3, 400 MHz) : 7.36-7.18 (m, 5H), 6.75 (bs, 1H), 3.86 (dd, J=11.6 Hz, 4.8 Hz, 1H), 3.74 (dd, J=11.2 Hz, 4.8 Hz, 1H), 3.54 (bs, 1H), 3.47 (dd, J=3.6 Hz, 1.2 Hz, 1H), 3.25 (dd, J=14.4 Hz, 3.2 Hz, 1H), 3.10 (m, 1H), 2.52 (dd, J=14.4 Hz, 10.4 Hz, 1H), 2.40 (m, 1H), 1.80 (bs, 1H), 1.02 (s, 9H), 0.90 (s, 9H), 0.10 (s, 3H), 0.08 (s, 3H) ppm. .sup.13C NMR (CDCl.sub.3, 100 MHz) : 184.8, 169.0, 138.5, 129.7, 128.9, 127.0, 63.9, 57.5, 53.6, 50.7, 44.6, 39.0, 35.5, 28.5, 26.1, 18.6, 5.0 ppm. HRMS (ESI) [MH].sup.+ calcd. 446.2833. found 446.2831.
(3R,5R,6R,7S)-3-benzyl-N-tert-butyl-7-((tert-butyldimethylsilyloxy)methyl)-2-oxo-1,4-diazabicyclo[4.1.0]heptane-5-carboxamide (2)
(106) .sup.1H NMR (CDCl.sub.3, 400 MHz) : 7.35-7.20 (m, 5H), 6.32 (bs, 1H), 3.86 (m, 1H), 3.73 (dd, J=11.5 Hz, 4.2 Hz, 1H), 3.86 (bs, 1H), 3.73 (dd, J=11.5 Hz, 4.2 Hz, 1H), 3.25 (dd, J=14.4 Hz, 3.2 Hz, 1H), 3.10 (m, 1H), 2.52 (dd, J=14.4 Hz, 10.4 Hz, 1H), 2.40 (m, 1H), 1.80 (bs, 1H), 1.31 (s, 9H), 0.90 (s, 9H), 0.10 (s, 3H), 0.08 (s, 3H) ppm. .sup.13C NMR (CDCl.sub.3, 75 MHz) : 184.0, 169.1, 136.2, 129.2, 129.1, 127.3, 63.0, 59.5, 51.4, 49.6, 42.2, 38.7, 36.0, 28.8, 26.1, 18.7, 5.1 ppm. MS (ESI) [MH].sup.+ calcd. 446.3. found 446.3.
(3S,5R,6R,7S)N-tert-butyl-7-((tert-butyldimethylsilyloxy)methyl)-3-methyl-2-oxo-1,4-diazabicyclo[4.1.0]heptane-5-carboxamide (3)
(107) .sup.1H NMR (CDCl.sub.3, 400 MHz) : 7.42 (s, NH), 3.88 (dd, J=11.2 Hz, 4.8 Hz, 1H), 3.70 (dd, J=11.2 Hz, 5.2 Hz, 1H), 3.55 (d, J=0.8 Hz, 1H), 3.46 (dd, J=3.6 Hz, 1.6 Hz, 1H), 3.07 (m, 1H), 2.36 (td, J=4.8 Hz, 3.6 Hz, 1H), 1.8 (bs, NH), 1.39 (s, 9H), 1.23 (d, J=6.8 Hz, 3H), 0.90 (s, 9H), 0.09 (s, 3H), 0.08 (s, 3H) ppm. .sup.13C NMR (CDCl.sub.3, 100 MHz) : 185.5, 169.3, 63.9, 53.8, 51.2, 51.0, 44.5, 39.3, 28.9, 26.1, 16.6, 15.0, 5.0 ppm. HRMS (ESI) [MH].sup.+ calcd. 370.2520. found 370.2520.
(1S,3aS,8R,8aS)N-tert-butyl-1-((tert-butyldimethylsilyloxy)methyl)-3-oxooctahydroazirino[1,2-a]pyrrolo[1,2-d]pyrazine-8-carboxamide (4)
(108) .sup.1H NMR (CDCl.sub.3, 400 MHz) : 6.17 (s, 1H), 3.76 (dd, J=11.6 Hz, 4.4 Hz, 1H), 3.67 (dd, J=11.6 Hz, 4.8 Hz, 1H), 3.70 (d, J=6.2 Hz, 1H), 3.12 (dt, J=4.8 Hz, 9.2 Hz, 1H), 2.97 (dd, J=6.4 Hz, 3.6 Hz, 1H), 2.93-2.89 (m, 1H), 2.60 (q, J=4.4 Hz, 1H), 2.25-2.05 (m, 2H), 1.90-1.75 (m, 3H), 1.70-1.51 (m, 1H), 1.48 (s, 9H), 0.85 (s, 9H), 0.09 (s, 3H), 0.08 (s, 3H) ppm. .sup.13C NMR (CDCl.sub.3, 100 MHz) : 183.0, 168.6, 65.0, 63.4, 63.2, 54.6, 51.3, 43.4, 41.0, 29.0, 26.1, 22.2, 22.0, 18.9, 5.1 ppm. HRMS (ESI) [MH].sup.+ calcd. 396.2676. found 396.2656.
(3R,5S,6S,7S)N-tert-butyl-3-(3-guanidinopropyl)-7-isobutyl-2-oxo-1,4 diazabicyclo[4.1.0]heptane-5-carboxamide (5)
(109) .sup.1H NMR (D.sub.2O, 400 MHz) : 3.59 (t, J=5.6 Hz, 1H), 3.46 (m, 1H), 3.13 (m, 1H), 2.94 (m, 1H), 3.37 (m, 1H), 1.78-1.44 (m, 8H), 1.22 (s, 9H), 0.83 (d, J=2.8 Hz, 3H), 0.81 (d, J=2.8 Hz, 3H) ppm. .sup.13H NMR (D.sub.2O, 400 MHz) : 186.7, 172.0, 156.9, 54.9, 54.5, 54.0, 44.5, 42.8, 28.7, 27.9, 26.5, 25.7, 25.0, 24.1, 22.2, 21.5 ppm. MS (ESI) [MH].sup.+ calcd. 367.3. found 367.2. and 184.1 for [M+2H].sup.2+/2.
(3S,5S,6S,7S)-3-(4-aminobutyl)-N-tert-butyl-7-isobutyl-2-oxo-1,4-diazabicyclo[4.1.0]heptane-5-carboxamide (6)
(110) NMR (D.sub.2O with 10% v/v CD.sub.3COOD, 400 MHz) : 5.14 (ddd, J=9.6 Hz, 4.4 Hz, 2.4 Hz, 1H), 4.12 (d, J=2.4 Hz, 1H), 3.07 (dd, J=7.6 Hz, 4.4 Hz, 1H), 2.85 (t, J=7.6 Hz, 2H), 1.82-1.46 (m, 10H), 1.32 (s, 9H), 0.83 (d, J=6.8 Hz, 3H), 0.81 (d, J=6.8 Hz, 3H) ppm. .sup.13C NMR (D.sub.2O with 10% v/v CD.sub.3COOD, 100 MHz) : 178.0, 173.8, 97.2, 58.6, 54.5, 53.0, 40.6, 39.3, 29.3, 27.8, 27.6, 27.2, 26.8, 24.6, 22.2, 21.9, 20.8 ppm. MS (ESI) calcd. 339.3. found 339.2 and 170.1 for [M+2H].sup.2+/2.
(5R,6R,7S)N-tert-butyl-7-((tert-butyldimethylsilyloxy)methyl)-2-oxo-1,4-diazabicyclo[4.1.0]heptane-5-carboxamide (7)
(111) .sup.1H NMR (400 MHz, CDCl.sub.3) : 6.98 (s, 1H), 3.87 (dd, J=11.5, 4.5 Hz, 1H), 3.78 (dd, J=11.5, 4.5 Hz, 1H), 3.34-3.24 (m, 2H), 3.13 (d, J=4.6 Hz, 1H), 2.40 (td, J=4.5, 3.4 Hz, 1H), 2.17-1.96 (m, 1H), 1.43-1.36 (m, 9H), 0.94-0.86 (m, 9H), 0.13-0.05 (m, 6H). .sup.13C NMR (101 MHz, CDCl.sub.3) : 183.93, 168.37, 77.48, 77.16, 76.84, 63.02, 55.81, 51.25, 48.72, 45.27, 39.64, 28.87, 26.06, 18.55, 0.15, 5.09, 5.12.
2-((3S,5R,6R,7R)-5-(tert-butylcarbamoyl)-7-isobutyl-2-oxo-1,4-diazabicyclo[4.1.0]heptan-3-yl)acetic acid (8)
(112) .sup.1H NMR (400 MHz, CD.sub.3OD) : 4.69-4.58 (m, 1H), 3.92 (d, J=2.0 Hz, 1H), 3.76 (dd, J=9.8, 3.6 Hz, 1H), 3.66 (dd, J=7.8, 4.0 Hz, 1H), 2.90 (dd, J=17.5, 3.6 Hz, 1H), 2.81 (dd, J=16.7, 4.0 Hz, 1H), 2.69-2.53 (m, 2H), 1.36-1.32 (m, 9H), 0.98 (dt, J=8.5, 4.2 Hz, 6H). .sup.13C NMR (101 MHz, CD.sub.3OD) 188.21, 172.40, 158.66, 158.62, 56.05, 55.25, 55.23, 51.95, 44.17, 43.55, 43.35, 42.55, 42.39, 42.16, 41.98, 29.85, 28.90, 28.85, 28.10, 27.37, 27.16, 26.85, 26.51, 23.19, 22.63, 21.05.
(3R,5R,6R,7S)N-(tert-butyl)-7-(((tert-butyldimethylsilyl)oxy)methyl)-3-(mercaptomethyl)-2-oxo-1,4-diazabicyclo[4.1.0]heptane-5-carboxamide (9)
(113) Note: proper spectroscopic characterization of this compound is difficult as spectra are characterized by extremely broad peaks. This is attributed to inherent instability of 9 due to the presence of nucleophilic thiol and its susceptibility to oxidation and aziridine ring-opening.
(114) .sup.1H NMR (CDCl.sub.3, 400 MHz) : 3.47 (s, 1H), 3.41 (d, J=6.5 Hz, 1H), 4.35-2.42 (m, 7H), 1.70-1.42 (bs, 10H), 0.90 (s, 9H), 0.10 (s, 3H), 0.08 (s, 3H) ppm. MS (ESI) [MH].sup.+ calcd. 402.6. found 402.2.
(3S,5R,6R,7S)-3-((1H-imidazol-4-yl)methyl)-N-(tert-butyl)-7-(((tert-butyldimethylsilyl)oxy)methyl)-2-oxo-1,4-diazabicyclo[4.1.0]heptane-5-carboxamide (10)
(115) .sup.1H NMR (CDCl.sub.3, 400 MHz) : 7.56 (s, 1H), 7.25 (s, 1H), 6.87 (s, 1H), 6.22 (s, 1H), 4.46 (m, 1H), 3.72 (dd, J=11.2 Hz, 4.8 Hz, 1H), 3.60 (m, 1H), 3.44 (dd, J=3.6 Hz, 1.5 Hz, 1H), 3.27 (dd, J=14.1 Hz, 3.1 Hz, 1H), 2.99 (dd, J=14.7 Hz, 4.2 Hz, 1H), 2.80 (dd, J=15.2 Hz, 8.1 Hz, 1H), 2.40 (m, 1H), 2.05 (bs, 1H), 1.36 (s, 9H), 0.87 (s, 9H), 0.07 (s, 3H), 0.05 (s, 3H) ppm. .sup.13C NMR (CDCl.sub.3, 100 MHz) : 185.4, 171.4, 169.5, 168.6, 135.1, 64.3, 62.7, 60.6, 54.7, 51.2, 44.5, 39.3, 28.9, 26.0, 18.4, 5.3 ppm. MS (ESI) [MH].sup.+ calcd. 436.6. found 436.2.
(3S,5R,6R,7S)N-(tert-butyl)-7-(((tert-butyldimethylsilyl)oxy)methyl)-3-(hydroxymethyl)-2-oxo-1,4-diazabicyclo[4.1.0]heptane-5-carboxamide (11)
(116) .sup.1H NMR (CDCl.sub.3, 400 MHz) : 6.80 (bs, 1H), 6.15 (bs, 1H), 4.02 (d, J=11.6 Hz, 1H), 3.85 (dd, J=13.3 Hz, 6.7 Hz, 1H), 3.76 (dd, J=14.2 Hz, 3.0 Hz, 1H), 3.68 (bs, 1H), 3.53 (d, J=5.0 Hz, 1H), 3.45 (dd, J=3.5 Hz, 2.4 Hz, 1H), 3.13 (m, 1H), 2.55 (m, 1H), 2.42 (m, 1H), 1.39 (s, 9H), 0.90 (s, 9H), 0.10 (s, 3H), 0.08 (s, 3H) ppm. .sup.13C NMR (CDCl.sub.3, 100 MHz) : 185.3, 168.9, 63.5, 61.4, 55.8, 53.9, 51.2, 44.9, 39.6, 28.9, 26.1, 18.6, 5.1 ppm. MS (ESI) [MH].sup.+ calcd. 386.6. found 386.2.
(1S,4S,6aS,11R,11 aS)N-tert-butyl-1-((tert-butyldimethylsilyloxy)methyl)-4-isobutyl-3,6-dioxodecahydro-1H-azirino[1,2-a]pyrrolo[1,2-d][1,4,7]triazonine-11-carboxamide (12)
(117) .sup.1H NMR (CD.sub.3OD, 400 MHz) : 4.47 (m, 1H), 4.14 (dd, J=8.4 Hz, 6 Hz, 1H), 4.07-3.99 (m, 2H), 3.84 (dd, J=12 Hz, 3.2 Hz, 1H), 3.32 (d, J=5.6 Hz, 1H), 3.26-3.15 (m, 2H), 2.42-2.30 (m, 2H), 1.92-1.78 (m, 3H), 1.74 (q, J=6.8 Hz, 2H), 1.59 (sept, J=6.8 Hz, 1H), 1.22 (s, 9H), 0.90 (d, J=6.8 Hz, 3H), 0.87 (d, J=6.4 Hz, 3H), 0.84 (s, 9H), 0.06 (s, 3H), 0.03 (s, 3H) ppm. .sup.13C NMR (CD.sub.3OD, 100 MHz) : 174.1, 164.1, 157.4, 86.5, 62.4, 60.7, 60.6, 57.7, 54.2, 52.1, 50.2, 39.6, 29.0, 25.8, 25.2, 25.1, 22.1, 21.0, 20.5, 17.9, 6.5 ppm. MS (ESI) calcd. 509.3. found 509.3.
(1S,4S,6aR,11S,11aR)N-tert-butyl-4-(3-guanidinopropyl)-1-isobutyl-3,6-dioxodecahydro-1H-azirino[1,2-a]pyrrolo[1,2-d][1,4,7]triazonine-11-carboxamide (13)
(118) .sup.1H NMR (CD.sub.3OD, 400 MHz) : 4.26 (t, J=5.6 Hz, 1H), 4.18 (dd, J=8.8 Hz, 2.4 Hz, 1H), 3.58 (dd, J=8.8 Hz, 5.2 Hz, 1H), 3.28-3.24 (m, 2H), 3.00 (d, J=5.6 Hz, 1H), 2.70 (dd, J=10.4 Hz, 6.4 Hz, 1H), 2.32-2.18 (m, 2H), 1.98-1.52 (m, 11H), 1.28 (s, 9H), 1.01 (d, J=6.4 Hz, 3H), 0.96 (d, J=6.4 Hz, 3H) ppm. .sup.13C NMR (CD.sub.3OD, 100 MHz) : 178.0, 160.8, 159.1, 157.4, 86.9, 64.3, 63.5, 60.2, 54.5, 53.7, 51.0, 42.6, 41.4, 29.4, 28.8, 26.1, 24.9, 24.5, 22.7, 21.3, 20.4 ppm. MS(ESI) [MH].sup.+ calcd. 464.3. found 464.3 and 232.6 for [M+2H].sup.2+/2.
2-((1S,4S,6aR,11S,11aR)-11-(tert-butylcarbamoyl)-1-isobutyl-3,6-dioxodecahydro-1H-azirino[1,2-a]pyrrolo[1,2-d][1,4,7]triazonin-4-yl)acetic acid (14)
(119) .sup.1H NMR (CD.sub.3OD, 400 MHz) : 4.46 (t, J=9.2 Hz, 1H), 4.34 (dd, J=8.4 Hz, 2.0 Hz, 1H), 3.81 (dd, J=8.4 Hz, 5.6 Hz, 1H), 3.38-3.30 (m, 1H), 3.02 (dd, J=17.2 Hz, 2.4 Hz, 1H), 2.85-2.73 (m, 1H), 2.57 (dd, J=17.2 Hz, 8.4 Hz, 1H), 2.45-2.32 (m, 2H), 2.05-1.62 (m, 7H), 1.58-1.48 (m, 2H), 1.27 (s, 9H), 1.01 (d, J=6.4 Hz, 3H), 0.98 (d, J=6.4 Hz, 3H) ppm. MS (ESI) [MH].sup.+ calcd. 423.3. found 423.2.
(1S,4S,9aS,14R,14aS)N-tert-butyl-1-((tert-butyldimethylsilyloxy)methyl)-4-isobutyl-3,6,9-trioxotetradecahydroazirino[1,2-a]pyrrolo[1,2-d][1,4,7,10]tetraazacyclododecine-14-carboxamide (15)
(120) .sup.1H NMR (CD.sub.3OD, 400 MHz) : 4.55 (ddd, J=8 Hz, 3.2 Hz, 1.6 Hz, 1H), 4.32 (dd, J=10.4 Hz, 4 Hz, 1H), 4.21 (dd, J=14 Hz, 6 Hz, 1H), 4.15-4.05 (m, 3H), 3.91 (dd, J=12 Hz, 3.2 Hz, 1H), 3.41 Hz (dd, J=6 Hz, 1H), 3.36-3.28 (m, 1H), 2.50-2.30 (m, 2H), 2.00-1.50 (m, 7H), 1.31 (s, 9H), 0.98-0.92 (m, 6H), 0.92 (s, 9H), 0.13 (s, 3H), 0.11 (s, 3H) ppm. .sup.13C NMR (CD.sub.3OD, 100 MHz) : 178.3, 166.4, 165.1, 157.6, 86.7, 62.7, 61.0, 60.1, 54.2, 53.7, 52.6, 50.5, 44.0, 41.6, 28.9, 25.6, 25.2, 25.1, 25.1, 22.6, 20.7, 20.5, 17.9, 6.5, 6.5 ppm. MS (ESI) [MH].sup.+ calcd. 566.4. found 566.4.
(1R,1aS,2R,6aS,12S,18S)-18-benzyl-N-tert-butyl-1,12-diisobutyl-7,10,13,16,19-pentaoxoicosahydroazirino[1,2-a]pyrrolo[1,2-d][1,4,7,10,13,16]hexaazacyclooctadecine-2-carboxamide (16)
(121) .sup.1H NMR (CDCl.sub.3, 500 MHz) : 7.82 (bs, NH), 7.40-7.20 (m, 5H), 7.18 (bs, NH), 7.05 (bs, NH), 6.81 (bs, 2 NH), 4.64 (q, J=6.8 Hz, 1H), 4.13 (dd, J=16 Hz, 7.6 Hz, 1H), 4.10-3.90 (m, 2H), 3.57 (dd, J=16 Hz, 4.8 Hz, 1H), 3.50-3.39 (m, 2H), 3.26 (dd, J=14 Hz, 5.6 Hz, 1H), 3.12 (dd, J=14 Hz, 7.2 Hz, 1H), 3.12-3.08 (m, 1H), 3.00-2.92 (m, 2H), 2.63 (t, J=3.6 Hz, 1H), 2.28-2.18 (m, 1H), 1.90-1.70 (m, 2H), 1.37-1.25 (m, 9H), 1.10-0.88 (m, 12H) ppm. .sup.13C NMR (CDCl.sub.3, 125 MHz) : 181.4, 176.1, 175.7, 172.1, 170.1, 169.2, 136.4, 130.1, 128.7, 126.8, 56.2, 63.0, 43.2, 42.5, 40.2, 38.6, 37.9, 31.2, 30.0, 29.8, 29.5, 28.8, 28.7, 27.2, 25.1, 24.9, 24.3, 23.3, 23.1, 22.8, 22.7, 22.6, 22.4, 22.3, 21.9, MS (ESI) [MH].sup.+ calcd. 682.4. found 682.4.
(1S,12aS,17R,17aR)N-tert-butyl-1-isobutyl-3,6,9,12-tetraoxohexadecahydro-1H-azirino[1,2-a]pyrrolo[1,2-d][1,4,7,10,13]pentaazacyclopentadecine-17-carboxamide (17)
(122) .sup.1H NMR (400 MHz, CDCl.sub.3) : 4.57-4.44 (m, 1H), 4.32-3.70 (m, 10H), 3.38 (d, J=5.4 Hz, 1H), 3.28 (s, 1H), 3.14 (dd, J=11.2, 5.6 Hz, 1H), 2.52-2.41 (m, 1H), 2.37 (d, J=6.9 Hz, 1H), 2.15-1.65 (m, 8H), 1.64-1.48 (m, 2H), 1.42-1.23 (m, 8H), 1.00 (dt, J=21.4, 7.4 Hz, 6H).
(123) .sup.13C NMR (100 MHz, CD.sub.3OD) : 176.18, 171.23, 168.97, 165.90, 159.24, 86.37, 65.77, 63.49, 61.48, 59.76, 55.41, 51.43, 47.38, 45.65, 45.23, 44.59, 44.43, 44.19, 43.41, 42.46, 29.85, 29.43, 28.97, 28.58, 28.46, 27.72, 27.13, 26.74, 25.68, 23.84, 23.68, 23.14, 23.06, 22.16, 21.62.
tert-butyl 2-41S,4S,7S,13S,15aS,20R,20aS)-20-(tert-butylcarbamoyl)-1-(((tert butyldimethylsilyl)oxy)methyl)-4-methyl-3,6,9,12,15-pentaoxo-13-(3-(3-((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)guanidino)propyl)icosahydroazirino[1,2-a]pyrrolo[1,2-d][1,4,7,10,13,16]hexaazacyclooctadecin-7-yl)acetate (18)
(124) .sup.1H NMR (400 MHz, CDCl.sub.3) : 7.84 (d, J=9.7 Hz, 2H), 7.58 (dd, J=18.1, 9.1 Hz, 2H), 7.07 (s, 1H), 6.41 (s, 2H), 5.68 (s, 1H), 4.85 (dq, J=13.7, 6.8 Hz, 1H), 4.70-4.58 (m, 1H), 4.35 (q, J=8.1 Hz, 1H), 4.17-4.07 (m, 1H), 3.97 (dd, J=14.2, 7.5 Hz, 2H), 3.74 (s, 1H), 3.69-3.57 (m, 1H), 3.56-3.42 (m, 2H), 3.42-3.13 (m, 4H), 2.96 (s, 2H), 2.93 (d, J=2.3 Hz, 1H), 2.89-2.76 (m, 4H), 2.66-2.53 (m, 7H), 2.42-2.15 (m, 2H), 2.10 (s, 3H), 2.07-1.98 (m, 1H), 1.87-1.55 (m, 8H), 1.46 (s, 6H), 1.44 (s, 9H), 1.34 (s, 9H), 0.87 (s, 9H), 0.09 (s, 3H), 0.05 (s, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3) : 187.07, 176.14, 174.46, 171.46, 171.04, 169.11, 166.93, 158.77, 156.39, 138.63, 133.66, 132.55, 124.65, 117.55, 105.24, 86.49, 82.64, 63.92, 63.84, 60.81, 57.07, 51.69, 51.31, 50.37, 49.56, 45.47, 43.51, 40.43, 37.55, 35.99, 30.28, 29.17, 28.83, 28.22, 26.11, 24.61, 19.49, 18.71, 18.19, 17.31, 12.69, 5.11, 5.44. MS (ESI) [MH].sup.+ calcd. 1104.6. found 1104.6.
tert-butyl (4-((1S,4S,7S,10S,13S,15aS,20R,20aS)-13-((R)-1-(tert-butoxy)ethyl)-20-(tert-butylcarbamoyl)-1-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-10-(2-(methylthio)ethyl)-3,6,9,12,15-pentaoxoicosahydroazirino[1,2-a]pyrrolo[1,2d][1,4,7,10,13,16]hexaazacyclooctadecin-7-yl)butyl)carbamate (19)
(125) .sup.1H NMR (400 MHz, CDCl.sub.3) : 7.83 (d, J=8.1 Hz, 1H), 7.77 (s, 1H), 7.50 (d, J=21.3 Hz, 1H), 7.35 (m, 2H), 7.08 (dd, J=15.7, 7.5 Hz, 1H), 6.90 (s, 1H), 6.82 (s, 1H), 4.98 (d, J=27.6 Hz, 1H), 4.84-4.67 (m, 1H), 4.60 (s, 1H), 4.40-4.30 (m, 1H), 4.16-3.93 (m, 4H), 3.82 (s, 2H), 3.77-3.69 (m, 3H), 3.67 (d, J=6.3 Hz, 1H), 3.45-3.28 (m, 2H), 3.27-2.99 (m, 6H), 3.00-2.86 (m, 2H), 2.84 (s, 1H), 2.78-2.55 (m, 3H), 2.45-2.19 (m, 5H), 2.12 (s, 4H), 2.06-1.97 (m, 3H), 1.89 (d, J=4.7 Hz, 7H), 1.71-1.55 (m, 3H), 1.54-1.39 (m, 21H), 1.36 (s, 13H), 1.31-1.26 (m, 12H), 1.06 (t, J=10.5 Hz, 4H), 0.93-0.82 (m, 14H), 0.06 (s, 3H), 0.05 (s, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3) : 182.99, 175.20, 171.94, 170.75, 170.37, 168.93, 156.44, 77.43, 75.69, 66.32, 64.35, 63.93, 61.77, 57.52, 56.58, 54.28, 42.68, 40.37, 31.21, 30.95, 30.08, 29.88, 29.36, 28.85, 28.66, 26.01, 24.92, 24.16, 18.91, 18.48, 17.89, 15.48, 5.25, 5.29. MS (ESI) [MH].sup.+ calcd. 983.6. found 983.6.
(1S,7S,10S,15aS,20R,20aS)-10-((1H-imidazol-5-yl)methyl)-7-(4-(tert-butoxy)benzyl)-N-(tert-butyl)-1-(((tert-butyldimethylsilyl)oxy)methyl)-3,6,9,12,15-pentaoxoicosahydroazirino[1,2-a]pyrrolo[1,2-d][1,4,7,10,13,16]hexaazacyclooctadecine-20-carboxamide (20)
(126) .sup.1H NMR (400 MHz, CDCl.sub.3) : 9.92 (s, 1H), 7.49 (d, J=4.8 Hz, 2H), 7.39-7.27 (m, 11H), 7.17-7.02 (m, 11H), 6.78 (d, J=8.4 Hz, 2H), 6.69 (s, 1H), 4.48 (p, J=6.9 Hz, 1H), 4.42-4.18 (m, 2H), 4.06-3.99 (m, 1H), 3.91 (dd, J=11.4, 3.8 Hz, 1H), 3.78 (dd, J=11.3, 4.4 Hz, 1H), 3.61 (dd, J=9.8, 4.8 Hz, 1H), 3.48-3.37 (m, 1H), 3.32-2.99 (m, 8H), 2.83 (dd, J=6.8, 3.4 Hz, 1H), 2.41-1.95 (m, 2H), 1.91-1.72 (m, 2H), 1.61 (s, 12H), 1.41 (s, 10H), 1.35-1.24 (m, 20H), 0.72 (s, 1H), 0.07 (s, 3H), 0.05 (s, 311). .sup.13C NMR (101 MHz, CDCl.sub.3) : 184.31, 175.20, 172.61, 170.67, 169.47, 168.12, 154.53, 142.31, 138.19, 136.81, 131.91, 129.97, 129.85, 128.43, 128.37, 120.09, 77.43, 63.49, 62.17, 61.55, 57.84, 54.97, 52.19, 51.15, 48.67, 43.36, 42.53, 41.75, 36.36, 31.84, 30.99, 29.92, 29.88, 29.44, 29.05, 28.67, 26.07, 24.49, 19.80, 18.56, 5.19. MS (ESI) [MH].sup.+ calcd. 866.5. found 866.5.
(1S,4S,7S,13S,15aS,20R,20aS)-13-benzyl-N-(tert-butyl)-1-(((tert-butyldimethylsilyl) oxy)methyl)-4-methyl-3,6,9,12,15-pentaoxo-7-(2-oxo-2-(tritylamino)ethyl) icosahydroazirino[1,2-a]pyrrolo[1,2-d][1,4,7,10,13,16]hexaazacyclooctadecine-20-carboxamide (21)
(127) .sup.1H NMR (400 MHz, CDCl.sub.3) : 7.94 (t, J=9.8 Hz, 2H), 7.73 (d, J=8.7 Hz, 1H), 7.51 (t, J=6.7 Hz, 1H), 7.34-7.11 (m, 23H), 6.79 (s, 1H), 4.92-4.72 (m, 2H), 4.59 (dd, J=16.3, 8.6 Hz, 1H), 4.25 (d, J=11.5 Hz, 1H), 4.01 (d, J=10.7 Hz, 1H), 3.82 (dd, J=14.2, 7.6 Hz, 1H), 3.73 (s, 1H), 3.59-3.51 (m, 2H), 3.48-3.24 (m, 3H), 3.09 (dd, J=13.8, 8.9 Hz, 1H), 2.96-2.68 (m, 6H), 2.59 (dd, J=15.8, 3.6 Hz, 1H), 2.45-2.08 (m, 2H), 2.07-1.96 (m, 1H), 1.62 (s, 5H), 1.52-1.17 (m, 18H), 0.94 (s, 9H), 0.15 (s, 3H), 0.13 (s, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3) : 184.06, 175.64, 173.93, 171.01, 170.49, 169.15, 167.20, 144.28, 136.98, 129.61, 128.81, 128.49, 128.30, 127.48, 126.75, 77.55, 77.23, 76.91, 71.17, 63.63, 60.72, 57.12, 52.96, 51.54, 50.58, 49.71, 49.27, 45.35, 44.53, 37.27, 36.80, 33.93, 30.22, 29.24, 26.24, 24.32, 18.78, 17.89, 0.22, 5.11, 5.34. MS (ESI) [MH].sup.+ calcd. 1027.5. found 1027.6.
(4S,10S,15aS,20R,20aS)-4-benzyl-N-(tert-butyl)-10-isobutyl-3,6,9,12,15-pentaoxoicosahydroazirino[1,2-a]pyrrolo[1,2-d][1,4,7,10,13,16]hexaazacyclooctadecine-20-carboxamide (22)
(128) .sup.1H NMR (CDCl.sub.3, 400 MHz) : 8.33 (bs, 1H), 8.17 (d, J=9.1 Hz, 1H), 8.03 (d, J=7.4 Hz, 1H), 7.54 (s, 1H), 7.21 (m, 5H), 6.04 (bs, 1H), 5.08 (bs, 1H), 4.58 (m, 1H), 3.87 (dd, J=8.1 Hz, 5.0 Hz, 1H), 3.80 (bs, 1H), 2.48 (dd, J=3.6 Hz, 1.8 Hz, 2H), 2.16 (m, 2H), 1.62-1.57 (m, 7H), 1.48 (m, 2H), 1.42 (m, 2H), 1.35 (m, 2H), 1.20 (m, 9H), 1.07 (m, 2H), 0.81 (m, 6H) ppm. MS (ESI) [MH].sup.+ calcd. 626.8. found 626.3 and 313.7 for [M+2H].sup.2+/2.
7-mmc conjugated 22 (23)
(129) Cyclic peptide 22 was reacted as a crude material to yield conjugated peptide 23. (see page 39).sup.1H NMR (300 MHz, DMSO) 8.95 (s, 1H), 8.68 (d, J=8.2 Hz, 1H), 8.32 (t, J=6.3 Hz, 1H), 7.68 (d, J=8.4 Hz, 1H), 7.51 (d, J=9.4 Hz, 1H), 7.42-7.08 (m, 5H), 6.46 (s, 1H), 6.31 (s, 1H), 4.47-4.15 (m, 3H), 3.73 (dd, J=20.5, 14.1 Hz, 3H), 3.45-3.33 (m, 2H), 3.17 (dd, J=20.9, 13.0 Hz, 2H), 2.98-2.64 (m, 4H), 2.40 (s, 2H), 2.22-2.10 (m, 1H), 1.91 (ddd, J=22.9, 13.4, 6.6 Hz, 2H), 1.79-1.40 (m, 5H), 1.30-1.15 (m, 9H), 0.93-0.74 (m, 6H). MS (ESI) [MH].sup.+ calcd. 818.4. found 818.3.
(130) HATU-Mediated Cyclization
(131) Advantageously, the cyclization reaction can be run at least at 0.002M concentration of the starting amino acid/peptide. Preferably the concentration is run between 0.002M to 0.2M.
(132) In another embodiment the process is run at a concentration of at least 0.1M of the starting amino acid/peptide. In another embodiment the process is run at a concentration of around 0.2M of the starting amino acid/peptide.
(133) Traditionally, prior macrocyclizations cannot be performed at such high concentrations. This was demonstrated using a direct comparison with HATU process which fails in that the major products observed are derived from oligomerization and polymerization.
(134) O-(7-Azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (HATU)-mediated cyclization of Pro-Gly-Gly-Gly was compared with the amino aldehyde/isocyanide cyclization method at 0.2 M. Upon completion, crude reaction mixtures were analyzed using liquid chromatography electrospray ionization mass spectrometry (LC-MS(ESI)) to assess the level of selectivity for the desired cyclic peptide.
(135) Mass spectrometry (MS)-analysis of the HATU reaction revealed that cyclodimerization predominated at 0.2M (
(136) Results from the MS peak analysis found 6 components.
(137) Component 1: Peak at Scan 27.4. Top ions are 655 566 923
(138) Component 2: Peak at Scan 28.4. Top ions are 982 744 714
(139) Component 3: Peak at Scan 29.9. Top ions are 235 848 538
(140) Component 4: Peak at Scan 30.9. Top ions are 559 827 560
(141) Component 5: Peak at Scan 31.9. Top ions are 752 931
(142) Component 6: Peak at Scan 32.7. Top ions are 537 295 446
(143) The crude LC-MS(ESI) analysis for the isocyanide/amino aldehyde mediated cyclization revealed a high level of selectivity for the desired cyclic peptide (m/z=479) at 0.2M (
(144) Results from the MS peak analysis found 2 components.
(145) Component 1: Peak at Scan 29.7. Top ions are 287 212 383 (Pro-Gly-Gly-Gly)
(146) Component 2: Peak at Scan 30.9. Top ions are 479 396 240 (cyclic peptide)
Example 2
Methods and Materials
(147) Method A: ring-opening of aziridine-containing cyclic peptide is conducted using an aromatic thiol as the nucleophile (Method A). In a screw-cap vial equipped with a magnetic stirring bar was added aziridine-containing cyclic peptide (0.06 mmol) and aromatic thiol (0.066 mmol) were added to 0.2 ml of degassed CHCl.sub.3. NEt.sub.3 (0.06 mmol) was then added to the solution and the reaction was stirred at room temperature for 3-4 hours. The reaction was then diluted with CH.sub.2Cl.sub.2, and washed twice with saturated aqueous NH.sub.4Cl (3 ml) followed by one wash with brine (3 ml). The organic layer was subsequently dried over Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. The crude ring-opened products were of high purity, but analytically pure samples were obtained following purification by flash column chromatography (5% MeOH in EtOAc).
(148) Method B: ring-opening of aziridine-containing cyclic peptide is carried out using an aliphatic thiol or imide as the nucleophile (Method B). In a screw-cap vial equipped with a magnetic stirring bar was added aziridine-containing cyclic peptide (0.06 mmol) and aliphatic thiol or imide (0.066 mmol) to 0.2 ml of degassed CHCl.sub.3. DBU (0.06 mmol) was then added to the solution and the reaction was stirred at room temperature for 3-4 hours. The reaction was then diluted with CH.sub.2Cl.sub.2, and washed twice with saturated aqueous NH.sub.4Cl (3 ml) followed by one wash with brine (3 ml). The organic layer was subsequently dried over Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. The crude ring-opened products were of high purity, but analytically pure samples were obtained following purification by flash column chromatography (5% MeOH in EtOAc).
(149) Results
(150) Side chains including, but not limited to, fluorescent substituents have been appended to the products of macrocyclization (Scheme 3).
(151) ##STR00026##
(152) The incorporation of an activated aziridine ring into the framework of a cyclic peptide provides a useful point for conjugation to various side-chains via nucleophilic ring-opening, a well developed methodology that has been demonstrated using nucleophilic biomolecules ranging from carbohydrates to biotin and farnesyl derivatives..sup.14 As an example, cyclic peptide conjugation strategy can be demonstrated through the nucleophilic ring-opening of an aziridine moiety with the widely used fluorescent tag 7-mercapto-4-methyl-coumarin (7-mmc) (Scheme 3). In another aspect, a late-stage synthesis of a thioester residue by ring-opening of the cyclic peptide with commercially available thiobenzoic acid can also be used. The reaction proceeded smoothly, delivering the ring opened product in 98% yield. The late-stage incorporation of the thioester functionality avoids steps typically required for its protection from both oxidation and electrophilic reagents. Many other amino acid side chains, both natural and unnatural, can be installed by similar ring-opening protocols, opening doors for conformational optimization of cyclic peptides at a late stage of synthesis. Other nucleophiles such as aliphatic thiols, acids, secondary amines such as morpholine, and imides worked equally well during ring-opening.
(153) In another aspect, cyclic peptides equipped with aziridine rings are subjected to ring-opening (
(154) These data suggest that strong ion pairing (pK.sub.a>1) between the base and nucleophile may result in decreased reactivity. The optimized conditions for ring-opening were found to proceed cleanly to afford the modified cyclic peptides in high purity following extractive work-up (
Example 3
(155) X-ray analysis of cyclic product 1 was conducted.
(156) Results
(157) ##STR00027##
(158) TABLE-US-00004 TABLE 4 Crystal data and structure refinement for k07227. Identification code k07227 Empirical formula C24H38N3O3Si Formula weight 445.67 Temperature 150(2) K Wavelength 0.71073 Crystal system Monoclinic Space group P 21 Unit cell dimensions a = 14.590(2) = 90. b = 12.326(2) = 94.718(7). c = 15.088(2) = 90. Volume 2704.2(7) .sup.3 Z 4 Density (calculated) 1.097 Mg/m.sup.3 Absorption coefficient 0.115 mm.sup.1 F(000) 968 Crystal size 0.32 0.10 0.08 mm.sup.3 Theta range for data collection 2.62 to 23.05. Index ranges 15 <= h <= 15, 13 <= k <= 12, 15 <= l <= 16 Reflections collected 10115 Independent reflections 3828 [R(int) = 0.1359] Completeness to theta = 23.05 95.9% Absorption correction Semi-empirical from equivalents Max. and min. transmission 1.000 and 0.770 Refinement method Full-matrix least-squares on F.sup.2 Data/restraints/parameters 3828/1/559 Goodness-of-fit on F.sup.2 1.044 Final R indices [I > 2 sigma(I)] R1 = 0.0982, wR2 = 0.2560 R indices (all data) R1 = 0.1715, wR2 = 0.3055 Largest diff. peak and hole 0.492 and 0.316 e .Math. .sup.3
(159) TABLE-US-00005 TABLE 5 Atomic coordinates (10.sup.4) and equivalent isotropic displacement parameters (.sup.2 10.sup.3) for k07227. U(eq) is defined as one third of the trace of the orthogonalized U.sup.ij tensor. x y z U(eq) Si(1A) 7245(3) 4768(4) 8396(3) 68(1) O(1A) 3310(7) 4696(12) 6317(8) 94(4) O(2A) 4778(6) 4429(8) 4729(7) 62(3) O(3A) 4070(7) 3807(11) 8551(7) 82(3) O(4A) 6881(6) 4731(9) 7332(6) 64(3) N(1A) 5222(7) 4298(9) 6198(7) 53(3) N(2A) 2735(8) 3545(12) 7673(8) 74(4) C(1A) 4577(10) 4187(12) 5473(9) 56(4) C(2A) 3624(9) 3854(14) 5700(9) 61(4) C(3A) 3884(8) 4758(14) 7161(9) 61(4) C(4A) 4916(9) 4610(11) 7073(9) 57(4) C(5A) 5474(9) 5392(12) 6570(9) 55(4) C(6A) 6464(10) 5591(15) 6823(10) 72(5) C(7A) 7044(16) 3423(18) 8820(12) 118(8) C(8A) 6568(12) 5820(20) 9009(13) 105(6) C(9A) 8501(12) 5097(14) 8489(14) 86(6) C(10A) 8607(13) 6160(30) 8022(19) 150(11) C(11A) 9045(13) 4350(30) 8043(17) 169(14) C(12A) 8846(14) 5210(20) 9454(15) 133(9) C(13A) 3590(10) 3973(17) 7871(11) 76(5) C(14A) 2268(10) 2780(16) 8263(10) 74(5) C(15A) 2819(13) 1767(18) 8419(13) 103(6) C(16A) 2093(11) 3415(16) 9107(10) 86(5) C(17A) 1325(12) 2540(20) 7748(12) 110(7) C(18A) 2957(9) 3707(15) 4897(9) 66(4) C(19A) 2151(10) 3006(18) 5091(10) 77(5) C(20A) 2294(13) 1877(19) 5232(12) 90(5) C(21A) 1548(17) 1230(20) 5361(14) 112(7) C(22A) 725(19) 1640(30) 5502(16) 128(9) C(23A) 525(13) 2770(30) 5385(13) 116(9) C(24A) 1270(12) 3480(20) 5217(11) 101(7) Si(1B) 8204(3) 2344(4) 2871(3) 68(1) O(1B) 6478(7) 2195(11) 6755(7) 86(3) O(2B) 4724(6) 1912(7) 5174(6) 53(2) O(3B) 8659(6) 1106(10) 6205(7) 75(3) O(4B) 7241(6) 2259(8) 3360(6) 66(3) N(1B) 6249(7) 1815(8) 4890(7) 48(3) N(2B) 7884(8) 963(11) 7449(9) 69(3) C(1B) 5494(10) 1740(12) 5431(9) 55(4) C(2B) 5816(9) 1389(12) 6398(9) 53(3) C(3B) 7344(9) 2232(14) 6288(8) 59(4) C(4B) 7165(9) 2098(12) 5256(9) 60(4) C(5B) 6589(10) 2897(14) 4693(11) 70(4) C(6B) 6790(11) 3147(14) 3790(11) 74(5) C(7B) 8766(12) 968(16) 3042(13) 97(6) C(8B) 8926(11) 3466(19) 3390(11) 98(6) C(9B) 7893(10) 2620(14) 1666(10) 71(5) C(10B) 7510(13) 3777(14) 1548(11) 85(5) C(11B) 7165(14) 1807(19) 1279(12) 103(6) C(12B) 8748(12) 2536(19) 1143(11) 100(6) C(13B) 8015(10) 1332(15) 6630(11) 69(4) C(14B) 8453(10) 163(13) 7952(11) 66(4) C(15B) 8439(13) 842(17) 7445(15) 111(7) C(16B) 9438(11) 564(17) 8135(13) 97(6) C(17B) 8040(11) 0(20) 8825(11) 103(7) C(18B) 5021(9) 1225(13) 6976(9) 61(4) C(19B) 5242(8) 526(15) 7787(10) 65(5) C(20B) 5337(10) 610(14) 7633(11) 65(4) C(21B) 5477(11) 1260(20) 8343(13) 101(7) C(22B) 5524(12) 940(20) 9202(18) 105(7) C(23B) 5439(11) 130(30) 9374(13) 100(7) C(24B) 5292(10) 878(19) 8643(11) 86(6)
(160) TABLE-US-00006 TABLE 6 Bond lengths [] and angles [] for k07227. Si(1A)O(4A) 1.649(10) Si(1A)C(7A) 1.81(2) Si(1A)C(9A) 1.871(18) Si(1A)C(8A) 1.91(2) O(1A)C(3A) 1.468(17) O(1A)C(2A) 1.491(18) O(2A)C(1A) 1.221(15) O(3A)C(13A) 1.211(17) O(4A)C(6A) 1.418(18) N(1A)C(1A) 1.390(17) N(1A)C(4A) 1.479(16) N(1A)C(5A) 1.494(18) N(2A)C(13A) 1.36(2) N(2A)C(14A) 1.50(2) C(1A)C(2A) 1.52(2) C(2A)C(18A) 1.502(19) C(3A)C(13A) 1.53(2) C(3A)C(4A) 1.533(18) C(4A)C(5A) 1.507(19) C(5A)C(6A) 1.48(2) C(9A)C(11A) 1.42(3) C(9A)C(10A) 1.50(3) C(9A)C(12A) 1.51(3) C(14A)C(15A) 1.49(3) C(14A)C(16A) 1.53(2) C(14A)C(17A) 1.55(2) C(18A)C(19A) 1.51(2) C(19A)C(20A) 1.42(3) C(19A)C(24A) 1.44(2) C(20A)C(21A) 1.38(3) C(21A)C(22A) 1.34(3) C(22A)C(23A) 1.44(4) C(23A)C(24A) 1.44(3) Si(1B)O(4B) 1.643(9) Si(1B)C(9B) 1.868(16) Si(1B)C(8B) 1.871(19) Si(1B)C(7B) 1.893(18) O(1B)C(2B) 1.457(18) O(1B)C(3B) 1.496(16) O(2B)C(1B) 1.177(15) O(3B)C(13B) 1.214(16) O(4B)C(6B) 1.457(18) N(1B)C(1B) 1.428(17) N(1B)C(4B) 1.446(17) N(1B)C(5B) 1.461(19) N(2B)C(13B) 1.344(19) N(2B)C(14B) 1.461(19) C(1B)C(2B) 1.56(2) C(2B)C(18B) 1.522(18) C(3B)C(13B) 1.54(2) C(3B)C(4B) 1.567(18) C(4B)C(5B) 1.51(2) C(5B)C(6B) 1.45(2) C(9B)C(12B) 1.53(2) C(9B)C(10B) 1.54(2) C(9B)C(11B) 1.54(2) C(14B)C(15B) 1.45(3) C(14B)C(17B) 1.51(2) C(14B)C(16B) 1.52(2) C(18B)C(19B) 1.51(2) C(19B)C(24B) 1.36(2) C(19B)C(20B) 1.43(2) C(20B)C(21B) 1.34(2) C(21B)C(22B) 1.35(3) C(22B)C(23B) 1.35(3) C(23B)C(24B) 1.44(3) O(4A)Si(1A)C(7A) 105.7(8) O(4A)Si(1A)C(9A) 108.3(7) C(7A)Si(1A)C(9A) 111.0(9) O(4A)Si(1A)C(8A) 110.6(8) C(7A)Si(1A)C(8A) 110.1(10) C(9A)Si(1A)C(8A) 111.0(9) C(3A)O(1A)C(2A) 113.3(11) C(6A)O(4A)Si(1A) 126.6(10) C(1A)N(1A)C(4A) 119.6(10) C(1A)N(1A)C(5A) 121.0(11) C(4A)N(1A)C(5A) 60.9(8) C(13A)N(2A)C(14A) 124.4(13) O(2A)C(1A)N(1A) 120.1(13) O(2A)C(1A)C(2A) 124.6(12) N(1A)C(1A)C(2A) 115.0(11) O(1A)C(2A)C(18A) 112.0(12) O(1A)C(2A)C(1A) 106.7(12) C(18A)C(2A)C(1A) 113.3(11) O(1A)C(3A)C(13A) 113.7(13) O(1A)C(3A)C(4A) 114.4(11) C(13A)C(3A)C(4A) 108.6(12) N(1A)C(4A)C(5A) 60.0(8) N(1A)C(4A)C(3A) 118.7(11) C(5A)C(4A)C(3A) 122.9(12) C(6A)C(5A)N(1A) 116.8(12) C(6A)C(5A)C(4A) 122.1(12) N(1A)C(5A)C(4A) 59.1(8) O(4A)C(6A)C(5A) 112.4(12) C(11A)C(9A)C(10A) 105(2) C(11A)C(9A)C(12A) 111.3(18) C(10A)C(9A)C(12A) 109(2) C(11A)C(9A)Si(1A) 113.9(16) C(10A)C(9A)Si(1A) 107.0(12) C(12A)C(9A)Si(1A) 109.9(13) O(3A)C(13A)N(2A) 125.1(16) O(3A)C(13A)C(3A) 121.6(14) N(2A)C(13A)C(3A) 113.2(14) C(15A)C(14A)N(2A) 110.8(13) C(15A)C(14A)C(16A) 114.8(16) N(2A)C(14A)C(16A) 106.7(15) C(15A)C(14A)C(17A) 111.4(17) N(2A)C(14A)C(17A) 104.4(13) C(16A)C(14A)C(17A) 108.1(14) C(2A)C(18A)C(19A) 112.2(12) C(20A)C(19A)C(24A) 120.2(19) C(20A)C(19A)C(18A) 118.9(16) C(24A)C(19A)C(18A) 121(2) C(21A)C(20A)C(19A) 119(2) C(22A)C(21A)C(20A) 122(3) C(21A)C(22A)C(23A) 122(3) C(22A)C(23A)C(24A) 118(2) C(23A)C(24A)C(19A) 118(2) O(4B)Si(1B)C(9B) 107.5(6) O(4B)Si(1B)C(8B) 109.4(7) C(9B)Si(1B)C(8B) 110.9(8) O(4B)Si(1B)C(7B) 105.0(7) C(9B)Si(1B)C(7B) 111.3(9) C(8B)Si(1B)C(7B) 112.5(9) C(2B)O(1B)C(3B) 114.0(11) C(6B)O(4B)Si(1B) 125.9(10) C(1B)N(1B)C(4B) 122.0(11) C(1B)N(1B)C(5B) 117.7(11) C(4B)N(1B)C(5B) 62.6(9) C(13B)N(2B)C(14B) 126.0(13) O(2B)C(1B)N(1B) 124.1(13) O(2B)C(1B)C(2B) 124.4(12) N(1B)C(1B)C(2B) 111.4(11) O(1B)C(2B)C(18B) 113.1(11) O(1B)C(2B)C(1B) 107.3(11) C(18B)C(2B)C(1B) 112.9(11) O(1B)C(3B)C(13B) 111.0(12) O(1B)C(3B)C(4B) 112.7(10) C(13B)C(3B)C(4B) 107.8(12) N(1B)C(4B)C(5B) 59.2(8) N(1B)C(4B)C(3B) 118.6(10) C(5B)C(4B)C(3B) 122.0(13) C(6B)C(5B)N(1B) 118.9(14) C(6B)C(5B)C(4B) 121.4(13) N(1B)C(5B)C(4B) 58.2(9) C(5B)C(6B)O(4B) 113.1(13) C(12B)C(9B)C(10B) 107.8(14) C(12B)C(9B)C(11B) 109.0(15) C(10B)C(9B)C(11B) 109.1(14) C(12B)C(9B)Si(1B) 110.2(11) C(10B)C(9B)Si(1B) 109.8(12) C(11B)C(9B)Si(1B) 110.8(12) O(3B)C(13B)N(2B) 125.6(15) O(3B)C(13B)C(3B) 119.1(14) N(2B)C(13B)C(3B) 114.7(13) C(15B)C(14B)N(2B) 108.6(13) C(15B)C(14B)C(17B) 110.9(16) N(2B)C(14B)C(17B) 107.0(13) C(15B)C(14B)C(16B) 110.2(15) N(2B)C(14B)C(16B) 111.3(13) C(17B)C(14B)C(16B) 108.9(15) C(19B)C(18B)C(2B) 114.7(11) C(24B)C(19B)C(20B) 118.0(16) C(24B)C(19B)C(18B) 125.4(18) C(20B)C(19B)C(18B) 116.5(14) C(21B)C(20B)C(19B) 117.9(18) C(20B)C(21B)C(22B) 125(3) C(23B)C(22B)C(21B) 119(2) C(22B)C(23B)C(24B) 119(2) C(19B)C(24B)C(23B) 121(2)
(161) TABLE-US-00007 TABLE 7 Anisotropic displacement parameters (.sup.2 10.sup.3) for k07227. The anisotropic displacement factor exponent takes the form: 2.sup.2[h.sup.2 a*.sup.2U.sup.11 + . . . + 2 h k a* b* U.sup.12] U.sup.11 U.sup.22 U.sup.33 U.sup.23 U.sup.13 U.sup.12 Si(1A) 59(2) 75(3) 67(3) 5(2) 2(2) 7(2) O(1A) 67(6) 116(10) 101(8) 29(8) 23(6) 1(7) O(2A) 66(6) 55(6) 64(7) 6(5) 11(5) 0(5) O(3A) 61(6) 118(10) 66(7) 14(7) 8(5) 7(6) O(4A) 55(5) 64(6) 76(6) 5(6) 11(4) 3(5) N(1A) 54(7) 54(7) 54(7) 6(6) 19(6) 1(6) N(2A) 51(7) 99(11) 74(8) 17(8) 12(6) 6(7) C(1A) 77(10) 49(9) 40(8) 1(7) 2(7) 3(7) C(2A) 60(9) 70(10) 52(8) 9(8) 3(7) 6(8) C(3A) 44(7) 70(10) 70(9) 7(9) 5(6) 7(7) C(4A) 61(8) 46(9) 65(9) 9(7) 7(7) 0(7) C(5A) 51(8) 48(9) 64(9) 4(7) 4(7) 4(7) C(6A) 62(10) 86(13) 69(10) 20(10) 18(8) 7(9) C(7A) 170(20) 110(17) 71(12) 20(12) 25(12) 17(16) C(8A) 83(12) 124(18) 109(14) 12(13) 23(10) 3(12) C(9A) 69(10) 59(11) 130(16) 44(11) 17(11) 11(9) C(10A) 47(11) 180(30) 220(30) 30(20) 10(13) 16(14) C(11A) 61(13) 300(50) 140(20) 60(20) 2(13) 13(19) C(12A) 101(14) 170(20) 122(17) 57(17) 40(13) 18(15) C(13A) 49(9) 122(15) 59(9) 5(10) 12(8) 2(10) C(14A) 58(9) 101(14) 64(10) 15(9) 17(7) 2(9) C(15A) 95(14) 103(16) 113(15) 37(13) 27(11) 15(12) C(16A) 85(11) 105(15) 68(11) 27(10) 17(8) 17(10) C(17A) 82(12) 150(20) 95(13) 10(14) 15(10) 60(13) C(18A) 63(9) 86(12) 51(8) 3(8) 8(7) 16(9) C(19A) 59(10) 122(18) 49(9) 5(10) 2(7) 2(10) C(20A) 97(13) 92(15) 85(12) 4(11) 31(10) 19(12) C(21A) 118(18) 121(19) 100(15) 8(13) 21(13) 48(16) C(22A) 95(19) 190(30) 102(17) 11(19) 0(13) 26(19) C(23A) 50(11) 210(30) 86(14) 9(17) 1(9) 1(16) C(24A) 63(12) 170(20) 68(11) 7(13) 7(8) 0(13) Si(1B) 60(3) 75(3) 70(3) 8(3) 13(2) 2(2) O(1B) 71(7) 106(9) 83(7) 14(7) 14(5) 3(7) O(2B) 51(6) 44(5) 65(6) 3(5) 3(4) 3(4) O(3B) 52(6) 99(9) 75(7) 4(6) 9(5) 9(6) O(4B) 69(6) 60(6) 71(6) 11(6) 24(5) 5(5) N(1B) 60(7) 36(6) 49(6) 1(5) 6(5) 13(5) N(2B) 50(7) 75(9) 84(9) 10(8) 13(6) 3(7) C(1B) 57(10) 48(9) 61(9) 17(7) 6(7) 4(7) C(2B) 45(8) 50(9) 65(9) 11(7) 4(6) 0(7) C(3B) 51(8) 72(10) 54(8) 1(8) 3(6) 5(8) C(4B) 47(8) 62(10) 73(9) 25(8) 17(7) 8(7) C(5B) 59(9) 65(11) 86(12) 6(9) 8(8) 9(8) C(6B) 82(11) 66(11) 77(11) 15(9) 21(8) 17(9) C(7B) 84(12) 87(14) 123(15) 34(12) 28(10) 36(11) C(8B) 64(10) 147(19) 84(12) 19(13) 10(8) 22(11) C(9B) 56(9) 78(13) 82(11) 10(9) 14(8) 12(8) C(10B) 113(13) 64(11) 77(11) 23(10) 11(9) 7(10) C(11B) 121(16) 104(16) 84(12) 14(12) 11(11) 22(13) C(12B) 97(13) 117(17) 89(12) 32(13) 36(10) 13(12) C(13B) 48(9) 85(12) 75(11) 3(10) 18(8) 9(8) C(14B) 66(10) 50(10) 81(11) 11(9) 3(8) 0(8) C(15B) 88(13) 88(17) 152(19) 37(15) 11(12) 29(11) C(16B) 62(10) 112(17) 116(15) 14(13) 1(10) 1(10) C(17B) 80(11) 150(20) 74(11) 55(13) 12(9) 23(12) C(18B) 48(8) 73(11) 61(9) 5(8) 7(6) 6(7) C(19B) 34(7) 105(15) 56(10) 14(10) 8(6) 16(8) C(20B) 67(10) 64(11) 64(10) 11(9) 3(7) 1(8) C(21B) 76(12) 150(20) 74(13) 14(16) 6(10) 11(12) C(22B) 67(12) 120(20) 120(20) 35(17) 13(11) 20(13) C(23B) 60(11) 180(30) 64(12) 9(16) 10(8) 26(14) C(24B) 59(10) 138(17) 63(11) 3(12) 14(8) 8(10)
(162) TABLE-US-00008 TABLE 8 Hydrogen coordinates (10.sup.4) and isotropic displacement parameters (.sup.2 10.sup.3) for k07227. x y z U (eq) H(2A) 2439 3731 7164 89 H(2AB) 3677 3148 6028 73 H(3AA) 3805 5507 7399 73 H(4AA) 5261 4254 7594 69 H(5AA) 5126 6016 6284 66 H(6AA) 6788 5688 6277 86 H(6AB) 6530 6272 7171 86 H(7AA) 7391 2890 8499 177 H(7AB) 6386 3254 8734 177 H(7AC) 7246 3395 9455 177 H(8AA) 5915 5621 8958 157 H(8AB) 6644 6535 8745 157 H(8AC) 6797 5834 9638 157 H(10A) 9261 6348 8041 225 H(10B) 8274 6725 8319 225 H(10C) 8356 6093 7402 225 H(11A) 9691 4578 8109 254 H(11B) 8832 4327 7410 254 H(11C) 8989 3625 8301 254 H(12A) 9506 5375 9499 199 H(12B) 8742 4534 9768 199 H(12C) 8516 5804 9723 199 H(15A) 3407 1942 8750 154 H(15B) 2933 1440 7847 154 H(15C) 2478 1254 8764 154 H(16A) 2680 3561 9449 128 H(16B) 1699 2986 9469 128 H(16C) 1788 4103 8942 128 H(17A) 1421 2134 7206 166 H(17B) 1014 3229 7588 166 H(17C) 944 2115 8123 166 H(18A) 3279 3371 4415 80 H(18B) 2729 4426 4688 80 H(20A) 2894 1576 5238 108 H(21A) 1622 461 5349 135 H(22A) 261 1165 5684 154 H(23A) 81 3040 5417 140 H(24A) 1185 4248 5190 122 H(2B) 7412 1227 7705 83 H(2BB) 6146 681 6365 64 H(3BA) 7647 2950 6413 71 H(4BA) 7677 1752 4955 72 H(5BA) 6321 3505 5026 84 H(6BA) 7190 3796 3799 89 H(6BB) 6209 3326 3435 89 H(7BA) 8930 851 3678 146 H(7BB) 9322 938 2721 146 H(7BC) 8336 402 2817 146 H(8BA) 8613 4161 3276 147 H(8BB) 9522 3478 3134 147 H(8BC) 9023 3346 4033 147 H(10D) 7339 3913 916 127 H(10E) 7981 4300 1769 127 H(10F) 6967 3855 1883 127 H(11D) 7007 1971 649 154 H(11E) 6613 1863 1604 154 H(11F) 7412 1069 1336 154 H(12D) 8576 2679 512 149 H(12E) 9009 1805 1211 149 H(12F) 9206 3070 1372 149 H(15D) 8712 718 6882 166 H(15E) 7802 1087 7323 166 H(15F) 8792 1398 7788 166 H(16D) 9719 649 7571 146 H(16E) 9792 36 8509 146 H(16F) 9437 1264 8442 146 H(17D) 7413 280 8715 154 H(17E) 8026 694 9140 154 H(17F) 8414 521 9189 154 H(18C) 4814 1945 7173 73 H(18D) 4502 892 6609 73 H(20B) 5304 894 7046 78 H(21B) 5548 2016 8234 121 H(22B) 5615 1446 9672 126 H(23B) 5475 386 9970 120 H(24B) 5229 1630 8761 104
(163) Although preferred embodiments of the invention have been described herein, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims. All references mentioned herein are incorporated by reference in their entirety.
REFERENCES
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